Adeno-associated virus virions with variant capsid and methods of use thereof

ABSTRACT

The present disclosure provides adeno-associated virus (AAV) virions with altered capsid protein, where the AAV virions exhibit greater infectivity of retinal cells compared to wild-type AAV. The present disclosure further provides methods of delivering a gene product to a retinal cell in an individual, and methods of treating ocular disease.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No.R21EY016994 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

The pathologies of numerous retinal degenerative diseases can beattributed to a multitude of genetic factors, and individualizedtreatment options for afflicted patients are limited andcost-inefficient. Gene delivery of secretable neuroprotective factors toMüller cells, a type of retinal glia that contacts all classes ofretinal neurons, represents an ideal approach to mediate protection ofthe entire retina. Vehicles such as adeno-associated viral vector (AAV)are currently in use for the delivery of gene products. Although severalnaturally occurring AAV variants have been isolated with a variety oftropisms, or cellular specificities, these vectors inefficiently infectMüller cells via intravitreal injection.

AAV belongs to the Parvoviridae family and Dependovirus genus, whosemembers require co-infection with a helper virus such as adenovirus topromote replication, and AAV establishes a latent infection in theabsence of a helper. Virions composed of a 25 nm icosahedral capsidencompassing a 4.9 kb single-stranded DNA genome with two open readingframes: rep and cap. The non-structural rep gene encodes four regulatoryproteins essential for viral replication, whereas cap encodes threestructural proteins (VP1-3) that assemble into a 60-mer capsid shell.This viral capsid mediates the ability of AAV vectors to overcome manyof the biological barriers of viral transduction—including cell surfacereceptor binding, endocytosis, intracellular trafficking, andunpackaging in the nucleus.

LITERATURE

U.S. Patent Publication No. 2005/0053922; U.S. Patent Publication No.2009/0202490; McGee et al. (2001) Mol. Ther. 4:622; Buch et al. (2006)Mol. Ther. 14:700; Gregory-Evans et al. (2009) Mol. Vis. 15:962; U.S.Patent Publication No. 2010/0172871; Klimczak, et al. (2009) PLoS One 4:e7467.

SUMMARY OF THE INVENTION

The present disclosure provides adeno-associated virus (AAV) virionswith altered capsid protein, where the AAV virions exhibit greaterinfectivity of retinal cells compared to wild-type AAV. The presentdisclosure further provides methods of delivering a gene product to aretinal cell in an individual, and methods of treating ocular disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts Müller glia in the retina.

FIGS. 2A-2I depict rShH10 expression following intravitreal injection inadult rat retina.

FIGS. 3A and 3B depict transduction specificity and efficiency of ShH10.

FIGS. 4A and 4B depict rShH10 expression in the whole retina followingintravitreal injection.

FIGS. 5A-5D depict retinal astrocyte infectivity of ShH10.

FIGS. 6A-6C depict in vitro characterization of ShH10.

FIGS. 7A-7C depict rAAV6 N451D expression following intravitrealinjection.

FIGS. 8A-8C depict amino acid sequences of wild-type and variant AAVcapsids.

FIG. 9 depicts heparin binding affinity of ShH10, AAV2, and AAV6.

FIGS. 10A-10D depict expression of ShH10.Y445F.scCAG-GFP in Müller cellsafter intravitreal injection.

FIG. 11 depicts enzyme-linked immunosorbent assay (ELISA) measurementsof human glial-derived neurotrophic factor (hGDNF) protein in retinalhomogenates 2 and 3 months following intravitreal delivery ofShH10.Y445F.scCAG-hGDNF.

FIGS. 12A-12F depict scatter plots of electroretinography (ERG)measurements following GDNF delivery to the eye using a rAAV of thepresent disclosure.

FIGS. 13A-13G depict measurements of outer nuclear layer thickness,inner plexiform layer thickness, and photoreceptor outer segment lengthalong the vertical meridian of the eye from the optic nerve head to theora serrate in rats at 3 months postinjection.

DEFINITIONS

“AAV” is an abbreviation for adeno-associated virus, and may be used torefer to the virus itself or derivatives thereof. The term covers allsubtypes and both naturally occurring and recombinant forms, exceptwhere required otherwise. The abbreviation “rAAV” refers to recombinantadeno-associated virus, also referred to as a recombinant AAV vector (or“rAAV vector”). The term “AAV” includes AAV type 1 (AAV-1), AAV type 2(AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAVtype 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9(AAV-9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV,non-primate AAV, and ovine AAV. “Primate AAV” refers to AAV that infectprimates, “non-primate AAV” refers to AAV that infect non-primatemammals, “bovine AAV” refers to AAV that infect bovine mammals, etc.

An “rAAV vector” as used herein refers to an AAV vector comprising apolynucleotide sequence not of AAV origin (i.e., a polynucleotideheterologous to AAV), typically a sequence of interest for the genetictransformation of a cell. In general, the heterologous polynucleotide isflanked by at least one, and generally by two AAV inverted terminalrepeat sequences (ITRs). The term rAAV vector encompasses both rAAVvector particles and rAAV vector plasmids.

An “AAV virus” or “AAV viral particle” or “rAAV vector particle” refersto a viral particle composed of at least one AAV capsid protein(typically by all of the capsid proteins of a wild-type AAV) and anencapsidated polynucleotide rAAV vector. If the particle comprises aheterologous polynucleotide (i.e. a polynucleotide other than awild-type AAV genome, such as a transgene to be delivered to a mammaliancell), it is typically referred to as an “rAAV vector particle” orsimply an “rAAV vector”. Thus, production of rAAV particle necessarilyincludes production of rAAV vector, as such a vector is contained withinan rAAV particle.

“Packaging” refers to a series of intracellular events that result inthe assembly and encapsidation of an AAV particle.

AAV “rep” and “cap” genes refer to polynucleotide sequences encodingreplication and encapsidation proteins of adeno-associated virus. AAVrep and cap are referred to herein as AAV “packaging genes.”

A “helper virus” for AAV refers to a virus that allows AAV (e.g.wild-type AAV) to be replicated and packaged by a mammalian cell. Avariety of such helper viruses for AAV are known in the art, includingadenoviruses, herpesviruses and poxviruses such as vaccinia. Theadenoviruses encompass a number of different subgroups, althoughAdenovirus type 5 of subgroup C is most commonly used. Numerousadenoviruses of human, non-human mammalian and avian origin are knownand available from depositories such as the ATCC. Viruses of the herpesfamily include, for example, herpes simplex viruses (HSV) andEpstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) andpseudorabies viruses (PRV); which are also available from depositoriessuch as ATCC.

“Helper virus function(s)” refers to function(s) encoded in a helpervirus genome which allow AAV replication and packaging (in conjunctionwith other requirements for replication and packaging described herein).As described herein, “helper virus function” may be provided in a numberof ways, including by providing helper virus or providing, for example,polynucleotide sequences encoding the requisite function(s) to aproducer cell in trans.

An “infectious” virus or viral particle is one that comprises apolynucleotide component which it is capable of delivering into a cellfor which the viral species is tropic. The term does not necessarilyimply any replication capacity of the virus. As used herein, an“infectious” virus or viral particle is one that can access a targetcell, can infect a target cell, and can express a heterologous nucleicacid in a target cell. Thus, “infectivity” refers to the ability of aviral particle to access a target cell, infect a target cell, andexpress a heterologous nucleic acid in a target cell. Infectivity canrefer to in vitro infectivity or in vivo infectivity. Assays forcounting infectious viral particles are described elsewhere in thisdisclosure and in the art. Viral infectivity can be expressed as theratio of infectious viral particles to total viral particles. Totalviral particles can be expressed as the number of viral genome copies.The ability of a viral particle to express a heterologous nucleic acidin a cell can be referred to as “transduction.” The ability of a viralparticle to express a heterologous nucleic acid in a cell can be assayedusing a number of techniques, including assessment of a marker gene,such as a green fluorescent protein (GFP) assay (e.g., where the viruscomprises a nucleotide sequence encoding GFP), where GFP is produced ina cell infected with the viral particle and is detected and/or measured;or the measurement of a produced protein, for example by anenzyme-linked immunosorbent assay (ELISA).

A “replication-competent” virus (e.g. a replication-competent AAV)refers to a phenotypically wild-type virus that is infectious, and isalso capable of being replicated in an infected cell (i.e. in thepresence of a helper virus or helper virus functions). In the case ofAAV, replication competence generally requires the presence offunctional AAV packaging genes. In general, rAAV vectors as describedherein are replication-incompetent in mammalian cells (especially inhuman cells) by virtue of the lack of one or more AAV packaging genes.Typically, such rAAV vectors lack any AAV packaging gene sequences inorder to minimize the possibility that replication competent AAV aregenerated by recombination between AAV packaging genes and an incomingrAAV vector. In many embodiments, rAAV vector preparations as describedherein are those which contain few if any replication competent AAV(rcAAV, also referred to as RCA) (e.g., less than about 1 rcAAV per 10²rAAV particles, less than about 1 rcAAV per 10⁴ rAAV particles, lessthan about 1 rcAAV per 10⁸ rAAV particles, less than about 1 rcAAV per10¹² rAAV particles, or no rcAAV).

The term “polynucleotide” refers to a polymeric form of nucleotides ofany length, including deoxyribonucleotides or ribonucleotides, oranalogs thereof. A polynucleotide may comprise modified nucleotides,such as methylated nucleotides and nucleotide analogs, and may beinterrupted by non-nucleotide components. If present, modifications tothe nucleotide structure may be imparted before or after assembly of thepolymer. The term polynucleotide, as used herein, refers interchangeablyto double- and single-stranded molecules. Unless otherwise specified orrequired, any embodiment of the invention described herein that is apolynucleotide encompasses both the double-stranded form and each of twocomplementary single-stranded forms known or predicted to make up thedouble-stranded form.

A polynucleotide or polypeptide has a certain percent “sequenceidentity” to another polynucleotide or polypeptide, meaning that, whenaligned, that percentage of bases or amino acids are the same whencomparing the two sequences. Sequence similarity can be determined in anumber of different manners. To determine sequence identity, sequencescan be aligned using the methods and computer programs, including BLAST,available over the world wide web at ncbi.nlm.nih.gov/BLAST/. Anotheralignment algorithm is FASTA, available in the Genetics Computing Group(GCG) package, from Madison, Wis., USA, a wholly owned subsidiary ofOxford Molecular Group, Inc. Other techniques for alignment aredescribed in Methods in Enzymology, vol. 266: Computer Methods forMacromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press,Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Ofparticular interest are alignment programs that permit gaps in thesequence. The Smith-Waterman is one type of algorithm that permits gapsin sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also,the GAP program using the Needleman and Wunsch alignment method can beutilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970)

Of interest is the BestFit program using the local homology algorithm ofSmith Waterman (Advances in Applied Mathematics 2: 482-489 (1981) todetermine sequence identity. The gap generation penalty will generallyrange from 1 to 5, usually 2 to 4 and in many embodiments will be 3. Thegap extension penalty will generally range from about 0.01 to 0.20 andin many instances will be 0.10. The program has default parametersdetermined by the sequences inputted to be compared. Preferably, thesequence identity is determined using the default parameters determinedby the program. This program is available also from Genetics ComputingGroup (GCG) package, from Madison, Wis., USA.

Another program of interest is the FastDB algorithm. FastDB is describedin Current Methods in Sequence Comparison and Analysis, MacromoleculeSequencing and Synthesis, Selected Methods and Applications, pp.127-149, 1988, Alan R. Liss, Inc. Percent sequence identity iscalculated by FastDB based upon the following parameters:

Mismatch Penalty: 1.00;

Gap Penalty: 1.00;

Gap Size Penalty: 0.33; and

Joining Penalty: 30.0.

A “gene” refers to a polynucleotide containing at least one open readingframe that is capable of encoding a particular protein after beingtranscribed and translated.

A “small interfering” or “short interfering RNA” or siRNA is a RNAduplex of nucleotides that is targeted to a gene interest (a “targetgene”). An “RNA duplex” refers to the structure formed by thecomplementary pairing between two regions of a RNA molecule. siRNA is“targeted” to a gene in that the nucleotide sequence of the duplexportion of the siRNA is complementary to a nucleotide sequence of thetargeted gene. In some embodiments, the length of the duplex of siRNAsis less than 30 nucleotides. In some embodiments, the duplex can be 29,28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11or 10 nucleotides in length. In some embodiments, the length of theduplex is 19-25 nucleotides in length. The RNA duplex portion of thesiRNA can be part of a hairpin structure. In addition to the duplexportion, the hairpin structure may contain a loop portion positionedbetween the two sequences that form the duplex. The loop can vary inlength. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13nucleotides in length. The hairpin structure can also contain 3′ or 5′overhang portions. In some embodiments, the overhang is a 3′ or a 5′overhang 0, 1, 2, 3, 4 or 5 nucleotides in length.

As used herein, the term “microRNA” refers to any type of interferingRNAs, including but not limited to, endogenous microRNAs and artificialmicroRNAs (e.g., synthetic miRNAs). Endogenous microRNAs are small RNAsnaturally encoded in the genome which are capable of modulating theproductive utilization of mRNA. An artificial microRNA can be any typeof RNA sequence, other than endogenous microRNA, which is capable ofmodulating the activity of an mRNA. A microRNA sequence can be an RNAmolecule composed of any one or more of these sequences. MicroRNA (or“miRNA”) sequences have been described in publications such as Lim, etal., 2003, Genes & Development, 17, 991-1008, Lim et al., 2003, Science,299, 1540, Lee and Ambrose, 2001, Science, 294, 862, Lau et al., 2001,Science 294, 858-861, Lagos-Quintana et al., 2002, Current Biology, 12,735-739, Lagos-Quintana et al., 2001, Science, 294, 853-857, andLagos-Quintana et al., 2003, RNA, 9, 175-179. Examples of microRNAsinclude any RNA that is a fragment of a larger RNA or is a miRNA, siRNA,stRNA, sncRNA, tncRNA, snoRNA, smRNA, shRNA, snRNA, or other smallnon-coding RNA. See, e.g., US Patent Applications 20050272923,20050266552, 20050142581, and 20050075492. A “microRNA precursor” (or“pre-miRNA”) refers to a nucleic acid having a stem-loop structure witha microRNA sequence incorporated therein. A “mature microRNA” (or“mature miRNA”) includes a microRNA that has been cleaved from amicroRNA precursor (a “pre-miRNA”), or that has been synthesized (e.g.,synthesized in a laboratory by cell-free synthesis), and has a length offrom about 19 nucleotides to about 27 nucleotides, e.g., a maturemicroRNA can have a length of 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt,25 nt, 26 nt, or 27 nt. A mature microRNA can bind to a target mRNA andinhibit translation of the target mRNA.

“Recombinant,” as applied to a polynucleotide means that thepolynucleotide is the product of various combinations of cloning,restriction or ligation steps, and other procedures that result in aconstruct that is distinct from a polynucleotide found in nature. Arecombinant virus is a viral particle comprising a recombinantpolynucleotide. The terms respectively include replicates of theoriginal polynucleotide construct and progeny of the original virusconstruct.

A “control element” or “control sequence” is a nucleotide sequenceinvolved in an interaction of molecules that contributes to thefunctional regulation of a polynucleotide, including replication,duplication, transcription, splicing, translation, or degradation of thepolynucleotide. The regulation may affect the frequency, speed, orspecificity of the process, and may be enhancing or inhibitory innature. Control elements known in the art include, for example,transcriptional regulatory sequences such as promoters and enhancers. Apromoter is a DNA region capable under certain conditions of binding RNApolymerase and initiating transcription of a coding region usuallylocated downstream (in the 3′ direction) from the promoter.

“Operatively linked” or “operably linked” refers to a juxtaposition ofgenetic elements, wherein the elements are in a relationship permittingthem to operate in the expected manner. For instance, a promoter isoperatively linked to a coding region if the promoter helps initiatetranscription of the coding sequence. There may be intervening residuesbetween the promoter and coding region so long as this functionalrelationship is maintained.

An “expression vector” is a vector comprising a region which encodes apolypeptide of interest, and is used for effecting the expression of theprotein in an intended target cell. An expression vector also comprisescontrol elements operatively linked to the encoding region to facilitateexpression of the protein in the target. The combination of controlelements and a gene or genes to which they are operably linked forexpression is sometimes referred to as an “expression cassette,” a largenumber of which are known and available in the art or can be readilyconstructed from components that are available in the art.

“Heterologous” means derived from a genotypically distinct entity fromthat of the rest of the entity to which it is being compared. Forexample, a polynucleotide introduced by genetic engineering techniquesinto a plasmid or vector derived from a different species is aheterologous polynucleotide. A promoter removed from its native codingsequence and operatively linked to a coding sequence with which it isnot naturally found linked is a heterologous promoter. Thus, forexample, an rAAV that includes a heterologous nucleic acid encoding aheterologous gene product is an rAAV that includes a nucleic acid notnormally included in a naturally-occurring, wild-type AAV, and theencoded heterologous gene product is a gene product not normally encodedby a naturally-occurring, wild-type AAV.

The terms “genetic alteration” and “genetic modification” (andgrammatical variants thereof), are used interchangeably herein to referto a process wherein a genetic element (e.g., a polynucleotide) isintroduced into a cell other than by mitosis or meiosis. The element maybe heterologous to the cell, or it may be an additional copy or improvedversion of an element already present in the cell. Genetic alterationmay be effected, for example, by transfecting a cell with a recombinantplasmid or other polynucleotide through any process known in the art,such as electroporation, calcium phosphate precipitation, or contactingwith a polynucleotide-liposome complex. Genetic alteration may also beeffected, for example, by transduction or infection with a DNA or RNAvirus or viral vector. Generally, the genetic element is introduced intoa chromosome or mini-chromosome in the cell; but any alteration thatchanges the phenotype and/or genotype of the cell and its progeny isincluded in this term.

A cell is said to be “stably” altered, transduced, genetically modified,or transformed with a genetic sequence if the sequence is available toperform its function during extended culture of the cell in vitro.Generally, such a cell is “heritably” altered (genetically modified) inthat a genetic alteration is introduced which is also inheritable byprogeny of the altered cell.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The terms also encompass an amino acid polymer that has beenmodified; for example, disulfide bond formation, glycosylation,lipidation, phosphorylation, or conjugation with a labeling component.Polypeptides such as anti-angiogenic polypeptides, neuroprotectivepolypeptides, and the like, when discussed in the context of deliveringa gene product to a mammalian subject, and compositions therefor, referto the respective intact polypeptide, or any fragment or geneticallyengineered derivative thereof, which retains the desired biochemicalfunction of the intact protein. Similarly, references to nucleic acidsencoding anti-angiogenic polypeptides, nucleic acids encodingneuroprotective polypeptides, and other such nucleic acids for use indelivery of a gene product to a mammalian subject (which may be referredto as “transgenes” to be delivered to a recipient cell), includepolynucleotides encoding the intact polypeptide or any fragment orgenetically engineered derivative possessing the desired biochemicalfunction.

An “isolated” plasmid, nucleic acid, vector, virus, virion, host cell,or other substance refers to a preparation of the substance devoid of atleast some of the other components that may also be present where thesubstance or a similar substance naturally occurs or is initiallyprepared from. Thus, for example, an isolated substance may be preparedby using a purification technique to enrich it from a source mixture.Enrichment can be measured on an absolute basis, such as weight pervolume of solution, or it can be measured in relation to a second,potentially interfering substance present in the source mixture.Increasing enrichments of the embodiments of this invention areincreasingly more isolated. An isolated plasmid, nucleic acid, vector,virus, host cell, or other substance is in some embodiments purified,e.g., from about 80% to about 90% pure, at least about 90% pure, atleast about 95% pure, at least about 98% pure, or at least about 99%, ormore, pure.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease or at risk of acquiring the diseasebut has not yet been diagnosed as having it; (b) inhibiting the disease,i.e., arresting its development; and (c) relieving the disease, i.e.,causing regression of the disease.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, human and non-human primates, including simians and humans;mammalian sport animals (e.g., horses); mammalian farm animals (e.g.,sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents(e.g., mice, rats, etc.).

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “arecombinant AAV virion” includes a plurality of such virions andreference to “the Müller cell” includes reference to one or more Müllercells and equivalents thereof known to those skilled in the art, and soforth. It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides adeno-associated virus (AAV) virionswith altered capsid protein, where the AAV virions exhibit greaterinfectivity of retinal cells compared to wild-type AAV. The presentdisclosure further provides methods of delivering a gene product to aretinal cell in an individual, and methods of treating ocular disease.

Recombinant Adeno-Associated Virus Virions

The present disclosure provides an infectious, recombinantadeno-associated virus (rAAV) virion comprising: a) a variant AAV capsidprotein, where the variant AAV capsid protein comprises at least oneamino acid difference (e.g., amino acid substitution, amino acidinsertion, amino acid deletion) relative to a corresponding parental AAVcapsid protein, and where the variant capsid protein confers increasedinfectivity of a retinal cell (e.g., a Müller glial cell (also referredto herein as a “Müller cell”)) compared to the infectivity of theretinal cell (e.g., Müller glial) cell by an AAV virion comprising thecorresponding parental AAV capsid protein, where the AAV capsid proteindoes not comprise an amino acid sequence present in a naturallyoccurring AAV capsid protein; and b) a heterologous nucleic acidcomprising a nucleotide sequence encoding a heterologous gene product.In some embodiments, the parental AAV capsid protein is a wild-type AAVcapsid. For example, in some embodiments, the parental AAV capsidprotein is wild-type AAV6 capsid protein. In some cases, the AAV capsidprotein comprises an amino acid sequence having at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to the ShH10 amino acidsequence depicted in FIGS. 8A-C, where the AAV capsid protein does notcomprise an amino acid sequence present in a naturally occurring AAVcapsid protein.

A subject rAAV virion exhibits at least 5-fold, at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, ormore than 50-fold, increased infectivity of a retinal cell compared tothe infectivity of the retinal cell by an AAV virion comprising thecorresponding parental AAV capsid protein (e.g., a wild-type AAV capsidprotein). For example, subject rAAV virion exhibits at least 5-fold, atleast 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, atleast 50-fold, or more than 50-fold, increased infectivity of a retinalcell compared to the infectivity of the retinal cell by an AAV virioncomprising a wild-type AAV6 capsid protein.

For example, in some cases, a subject rAAV virion exhibits at least5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least25-fold, at least 50-fold, or more than 50-fold, increased infectivityof a Müller glial cell compared to the infectivity of the Müller glialcell by an AAV virion comprising the corresponding parental AAV capsidprotein (e.g., a wild-type AAV capsid protein). For example, in somecases, a subject rAAV virion exhibits at least 5-fold, at least 10-fold,at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold,or more than 50-fold, increased infectivity of a Müller glial cellcompared to the infectivity of the Müller glial cell by an AAV virioncomprising a wild-type AAV6 capsid protein.

Without being bound to theory, increased infectivity of a retinal cellsuch as a Müller glial cell, when a subject rAAV virion is administeredvia intravitreal injection, may be due to altered interactions withnaturally-occurring structures in the eye, e.g., increased ability ofthe rAAV virion to cross the inner limiting membrane.

In some embodiments, a subject rAAV virion selectively infects a Müllerglial cell, e.g., a subject rAAV virion infects a Müller glial cell with5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or more than50-fold, specificity than a non-Müller glial cell present in the eye,e.g., a retinal ganglion cell.

In some cases, a subject rAAV virion, when introduced (e.g., viaintravitreal injection or other route of administration) into an eye ofan individual, provides for high level production of the heterologousgene product encoded by the rAAV in the eye. For example, a heterologouspolypeptide encoded by the rAAV can be produced in the eye at a level offrom about 1 μg to about 50 or greater than 50 μg. As another example, aheterologous polypeptide encoded by the rAAV can be produced in thevitreous fluid of the eye at a level of from about 100 pg/mL to about5000 pg/mL vitreous fluid, e.g., from about 100 pg/mL to about 500pg/mL, from about 500 pg/mL to about 1000 pg/mL, from about 1000 pg/mLto about 2000 pg/mL, from about 2000 pg/mL to about 3000 pg/mL, fromabout 3000 pg/mL to about 4000 pg/mL, or from about 4000 pg/mL to about5000 pg/mL. In some cases, a polypeptide encoded by the rAAV can beproduced in the vitreous fluid of the eye at a level of greater than5000 pg/mL vitreous fluid.

In some cases, a subject rAAV virion, when introduced (e.g., viaintravitreal injection or other route of administration) into an eye ofan individual, provides for production of the heterologous gene productencoded by the rAAV in at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 50% at least about 60%, at leastabout 70%, at least about 80%, or more than 80%, of the Müller cells inthe eye.

In some embodiments, a subject rAAV virion, when introduced (e.g., viaintravitreal injection) into an eye of an individual, provides forproduction of the heterologous gene product encoded by the rAAV for aperiod of time of from about 2 days to about 6 months, e.g., from about2 days to about 7 days, from about 1 week to about 4 weeks, from about 1month to about 2 months, or from about 2 months to about 6 months. Insome embodiments, a subject rAAV virion, when introduced (e.g., viaintravitreal injection) into an eye of an individual, provides forproduction of the heterologous gene product encoded by the rAAV for aperiod of time of more than 6 months, e.g., from about 6 months to 20years or more, or greater than 1 year, e.g., from about 6 months toabout 1 year, from about 1 year to about 2 years, from about 2 years toabout 5 years, from about 5 years to about 10 years, from about 10 yearsto about 15 years, from about 15 years to about 20 years, or more than20 years.

Heterologous Nucleic Acid

A subject rAAV virion comprises a heterologous nucleic acid comprising anucleotide sequence encoding a heterologous gene product, e.g., anucleic acid gene product or a polypeptide gene product. In someembodiments, the gene product is an interfering RNA (e.g., shRNA, siRNA,miRNA). In some embodiments, the gene product is an aptamer. The geneproduct can be a self-complementary nucleic acid. In some embodiments,the gene product is a polypeptide.

Nucleic Acid Gene Products

Suitable nucleic acid gene products include interfering RNA, antisenseRNA, ribozymes, and aptamers. Where the gene product is an interferingRNA (RNAi), suitable RNAi include RNAi that decrease the level of anangiogenic factor in a cell. For example, an RNAi can be a miRNA, anshRNA, or an siRNA that reduces the level of vascular endothelial growthfactor (VEGF) in a cell.

Where the gene product is an interfering RNA (RNAi), suitable RNAiinclude RNAi that decrease the level of an angiogenic factor in a cell.For example, an RNAi can be an shRNA or siRNA that reduces the level ofVEGF or VEGF receptor (VEGFR) in a cell. RNAi agents that target VEGFinclude, e.g., an RNAi described in U.S. Patent Publication No.2011/0224282. For example, an siRNA specific for VEGF-A, VEGFR1, orVEGFR2 would be suitable. Suitable nucleic acid gene products alsoinclude a ribozyme specific for VEGF-A, VEGFR1, or VEGFR2; an antisensespecific for VEGF-A, VEGFR1, or VEGFR2; siRNA specific for VEGF-A,VEGFR1, or VEGFR2; etc.

Also suitable as a gene product is an miRNA that reduces the level ofVEGF by regulating VEGF gene expression, e.g., throughpost-transcriptional repression or mRNA degradation. Examples ofsuitable miRNA include, e.g., miR-15b, miR-16, miR-20a, and miR-20b.See, e.g., Hua et al. (2006) PLoS ONE 1:e116.

Also suitable is an anti-VEGF aptamer (e.g., EYE001). For anti-VEGFaptamers, see, e.g., Ng et al. (2006) Nature Reviews Drug Discovery5:123; and U.S. Pat. Nos. 6,426,335; 6,168,778; 6,147,204; 6,051,698;and 6,011,020. For example, an aptamer directed against VEGF₁₆₅, theisoform primarily responsible for pathological ocular neovascularizationand vascular permeability, would be suitable.

Polypeptide Gene Products

Where the gene product is a polypeptide, exemplary polypeptides includeneuroprotective polypeptides and anti-angiogenic polypeptides. Suitablepolypeptides include, but are not limited to, glial derived neurotrophicfactor (GDNF), fibroblast growth factor 2 (FGF-2), nurturin, ciliaryneurotrophic factor (CNTF), nerve growth factor (NGF; e.g., nerve growthfactor-(3), brain derived neurotrophic factor (BDNF), neurotrophin-3(NT-3), neurotrophin-4 (NT-4), neurotrophin-6 (NT-6), epidermal growthfactor (EGF), pigment epithelium derived factor (PEDF), a Wntpolypeptide, soluble Flt-1, angiostatin, endostatin, an anti-VEGFantibody, a soluble VEGFR, and a member of the hedgehog family (sonichedgehog, indian hedgehog, and desert hedgehog, etc.).

GDNF can be synthesized in cells as a 211-amino acid residueprepropeptide that is processed to yield a dimeric protein composed oftwo 134-amino acid residue subunits. GDNF has been described amply inthe literature; see, e.g., Lin et al. (1993) Science 260:1130; Grimm etal. (1998) Hum. Mol. Genet. 7:1873; Airaksinen and Saarma (2002) NatureReviews 3:383; and Kyuno and Jones (2007) Gene Expr. Patterns 7:313.GDNF amino acid sequences are known; see, e.g., GenBank Accession Nos.NP_000505, NP_001177397; NP_001177398; and NP_954701. Suitable for useherein is a GDNF polypeptide having at least about 85%, at least about90%, at least about 95%, or 100%, amino acid sequence identity to acontiguous stretch of 134 amino acids of the amino acid sequence ofamino acids 78-211 of the sequence set forth in SEQ ID NO:10. Activefragments of GDNF are also suitable for use. In some embodiments, a GDNFpolypeptide has a length of from about 75 amino acids (aa) to about 100aa, from about 100 aa to about 134 aa, from about 134 aa to about 185aa, from about 185 aa to about 202 aa, from about 202 aa to about 211aa, or from about 211 aa to about 228 aa.

PEDF is an approximately 418-amino acid polypeptide that exhibits bothneurotrophic and anti-angiogenic properties. Steele et al. (1993) Proc.Natl. Acad. Sci. USA 90:1526. PEDF amino acid sequences are known; see,e.g., GenBank Accession No. NP_002606; and Steele et al. (1993) supra. Asuitable PEDF polypeptide can have at least about 85%, at least about90%, at least about 95%, or 100%, amino acid sequence identity to acontiguous stretch of from about 25 amino acids (aa) to about 35 aa,from about 35 aa to about 45 aa, from about 45 aa to about 50 aa, fromabout 50 aa to about 100 aa, from about 100 aa to about 200 aa, fromabout 200 aa to about 300 aa, from about 300 aa to about 400 aa, or fromabout 400 aa to 418 aa, of the amino acid sequence set forth in SEQ IDNO:11. In some cases, the PEDF polypeptide is an active fragment. Forexample, a fragment comprising amino acids 24-57 can exhibitanti-angiogenic properties (see, e.g., Amaral and Becerra (2010) Invest.Ophthalmol. Vis. Sci. 51:1318); and a fragment comprising amino acids58-101 can exhibit neurotrophic properties (see, e.g., Filleur et al.(2005) Cancer Res. 65:5144).

Anti-angiogenic polypeptides include, e.g., vascular endothelial growthfactor (VEGF) antagonists. Suitable VEGF antagonists include, but arenot limited to, inhibitors of VEGFR1 tyrosine kinase activity;inhibitors of VEGFR2 tyrosine kinase activity; an antibody to VEGF; anantibody to VEGFR1; an antibody to VEGFR2; a soluble VEGFR; and thelike. See, e.g., Takayama et al. (2000) Cancer Res. 60:2169-2177; Moriet al. (2000) Gene Ther. 7:1027-1033; and Mahasreshti et al. (2001)Clin. Cancer Res. 7:2057-2066; and U.S. Patent Publication No.20030181377. Antibodies specific for VEGF include, e.g., bevacizumab(AVASTIN™) and ranibizumab (also known as rhuFAb V2). Also suitable foruse are anti-angiogenic polypeptides such as endostatin, PEDF, andangiostatin.

Anti-angiogenic polypeptides include, e.g., recombinant polypeptidescomprising VEGF receptors. For example, a suitable anti-angiogenicpolypeptide would be the soluble form of the VEGFR-1, known as sFlt-1(Kendall et al. (1996) Biochem. Biophys. Res. Commun. 226:324). Suitableanti-angiogenic polypeptides include an immunoglobulin-like (Ig) domain2 of a first VEGF receptor (e.g., Flt1), alone or in combination with anIg domain 3 of a second VEGF receptor (e.g., Flk1 or Flt4); theanti-angiogenic polypeptide can also include a stabilization and/or amultimerization component. Such recombinant anti-angiogenic polypeptidesare described in, e.g., U.S. Pat. No. 7,521,049.

Anti-VEGF antibodies that are suitable as heterologous gene productsinclude single chain Fv (scFv) antibodies. See, e.g., U.S. Pat. Nos.7,758,859; and 7,740,844, for anti-VEGF antibodies.

Structural Features

A subject rAAV virion can comprise a variant AAV capsid protein thatdiffers in amino acid sequence by at least one amino acid from awild-type capsid protein. The amino acid difference(s) can be located ina solvent accessible site in the capsid, e.g., a solvent-accessibleloop. For example, the amino acid substitution(s) can be located in a GHloop in the AAV capsid protein. In some cases, the variant capsidprotein comprises an amino acid substitution at amino acid 451 and/or532, compared to the amino acid sequence of AAV6 capsid (SEQ ID NO:1),or the corresponding amino acid in a serotype other than AAV6. In somecases, the variant capsid protein comprises an amino acid substitutionat amino acid 319 and/or 451 and/or 532 and/or 642, compared to theamino acid sequence of AAV6 capsid (SEQ ID NO:1), or the correspondingamino acid in a serotype other than AAV6. In some cases, the variantcapsid protein comprises one or more of the following substitutionscompared to the amino acid sequence of AAV6 capsid (SEQ ID NO:1): I319V,N451D, D532N, and H642N.

A subject rAAV virion can comprise a variant AAV capsid protein thatcomprises an amino acid sequence having at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, at least about 99%,or 100%, amino acid sequence identity to the ShH10 amino acid sequencedepicted in FIGS. 8A-C and SEQ ID NO:3, where the variant AAV capsidprotein comprises from 1 to about 10 amino acid differences (e.g., aminoacid substitutions and/or amino acid insertions and/or amino aciddeletions) compared to the AAV6 capsid protein depicted in FIGS. 8A-Cand SEQ ID NO:1. The amino acid difference(s) can be located in asolvent accessible site in the capsid, e.g., a solvent-accessible loop.For example, the amino acid substitution(s) can be located in a GH loopin the AAV capsid protein. In some cases, the variant capsid proteincomprises an I319V substitution, an N451D substitution, a D532Nsubstitution, and an H642N substitution compared to the amino acidsequence of AAV6 capsid (SEQ ID NO:1).

For example, the variant capsid protein can comprise an amino acidsequence having at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the ShH10 amino acid sequence depicted in SEQ IDNO:3, where the variant capsid protein an I319V substitution, an N451Dsubstitution, a D532N substitution, and an H642N substitution comparedto the amino acid sequence of AAV6 capsid (SEQ ID NO:1).

In some embodiments, the variant capsid protein can comprise an aminoacid sequence that has at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to the ShH10 amino acidsequence depicted in FIGS. 8A-C and SEQ ID NO:3, and that includes anI319V substitution, relative to AAV6 capsid. For example, the variantcapsid protein can comprise an amino acid sequence that has at leastabout 95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the ShH10 amino acid sequence set forth in SEQ IDNO:8 (comprising an I319V substitution compared to SEQ ID NO:1).

In some embodiments, the variant capsid protein can comprise an aminoacid sequence that has at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to the ShH10 amino acidsequence depicted in FIGS. 8A-C and SEQ ID NO:3, and that includes anI319V substitution and an N451D substitution, relative to AAV6 capsid.For example, the variant capsid protein can comprise an amino acidsequence that has at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to the ShH10 amino acidsequence set forth in SEQ ID NO:6 (comprising an I319V and an N451Dsubstitution compared to SEQ ID NO:1).

In some embodiments, the variant capsid protein can comprise an aminoacid sequence that has at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to the ShH10 amino acidsequence depicted in FIGS. 8A-C and SEQ ID NO:3, and that includes anI319V substitution and a D532N substitution, relative to AAV6 capsid.For example, the variant capsid protein can comprise an amino acidsequence that has at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to the ShH10 amino acidsequence set forth in SEQ ID NO:9 (comprising an I319V and a D532Nsubstitution compared to SEQ ID NO:1).

In some embodiments, the variant capsid protein can comprise an aminoacid sequence that has at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to the ShH10 amino acidsequence depicted in FIGS. 8A-C and SEQ ID NO:3, and that includes anN451D substitution and a D532N substitution, relative to AAV6 capsid.For example, the variant capsid protein can comprise an amino acidsequence that has at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to the ShH10 amino acidsequence set forth in SEQ ID NO:7 (comprising an N451D and a D532Nsubstitution compared to SEQ ID NO:1).

In some embodiments, the variant capsid protein can comprise an aminoacid sequence that has at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to the ShH10 amino acidsequence depicted in FIGS. 8A-C and SEQ ID NO:3, and that includes anI319V substitution, an N451D substitution, and a D532N substitution,relative to AAV6 capsid. For example, the variant capsid protein cancomprise an amino acid sequence that has at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity tothe ShH10 amino acid sequence set forth in SEQ ID NO:5 (comprising anI319V, an N451D, and a D532N substitution compared to SEQ ID NO:1).

Control Elements

The heterologous nucleotide sequence can be operably linked to controlelements that direct the transcription or expression thereof in thenucleotide sequence in vivo. Such control elements can comprise controlsequences normally associated with the selected gene (e.g., endogenouscellular control elements). Alternatively, heterologous controlsequences can be employed. Useful heterologous control sequencesgenerally include those derived from sequences encoding mammalian orviral genes. Examples include, but are not limited to, the SV40 earlypromoter, mouse mammary tumor virus long terminal repeat (LTR) promoter;adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV)promoter, an endogenous cellular promoter that is heterologous to thegene of interest, a cytomegalovirus (CMV) promoter such as the CMVimmediate early promoter region (CMVIE), a rous sarcoma virus (RSV)promoter, synthetic promoters, hybrid promoters, and the like. Inaddition, sequences derived from nonviral genes, such as the murinemetallothionein gene, can also be used. Such promoter sequences arecommercially available from, e.g., Stratagene (San Diego, Calif.).

In some embodiments, a cell type-specific or a tissue-specific promoterwill be operably linked to the heterologous nucleic acid encoding theheterologous gene product, such that the gene product is producedselectively or preferentially in a particular cell type(s) or tissue(s).In some embodiments, an inducible promoter will be operably linked tothe heterologous nucleic acid.

Methods for Generating an rAAV Virion

An AAV expression vector which comprises a heterologous nucleic acid andwhich is used to generate an rAAV virion, can be constructed usingmethods that are well known in the art. See, e.g., Koerber et al. (2009)Mol. Ther. 17:2088; Koerber et al. (2008) Mol Ther. 16:1703-4709: U.S.Pat. Nos. 7,439,065, 6,951,758, and 6,491,907. For example, theheterologous sequence(s) can be directly inserted into an AAV genomewhich has had the major AAV open reading frames (“ORFs”) excisedtherefrom. Other portions of the AAV genome can also be deleted, so longas a sufficient portion of the ITRs remain to allow for replication andpackaging functions. Such constructs can be designed using techniqueswell known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and5,139,941; International Publication Nos. WO 92/01070 (published Jan.23, 1992) and WO 93/03769 (published Mar. 4, 1993); Lebkowski et al.(1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90(Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) CurrentOpinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Curr. TopicsMicrobiol. Immunol. 158:97-129; Kotin, R. M. (1994) Human Gene Therapy5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-169; and Zhou etal. (1994) J. Exp. Med. 179:1867-1875.

In order to produce rAAV virions, an AAV expression vector is introducedinto a suitable host cell using known techniques, such as bytransfection. A number of transfection techniques are generally known inthe art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook etal. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratories, New York, Davis et al. (1986) Basic Methods in MolecularBiology, Elsevier, and Chu et al. (1981) Gene 13:197. Particularlysuitable transfection methods include calcium phosphate co-precipitation(Graham et al. (1973) Virol. 52:456-467), direct micro-injection intocultured cells (Capecchi, M. R. (1980) Cell 22:479-488), electroporation(Shigekawa et al. (1988) BioTechnigues 6:742-751), liposome mediatedgene transfer (Mannino et al. (1988) BioTechniques 6:682-690),lipid-mediated transduction (Felgner et al. (1987) Proc. Natl. Acad.Sci. USA 84:7413-7417), and nucleic acid delivery using high-velocitymicroprojectiles (Klein et al. (1987) Nature 327:70-73).

Suitable host cells for producing rAAV virions include microorganisms,yeast cells, insect cells, and mammalian cells, that can be, or havebeen, used as recipients of a heterologous DNA molecule. The termincludes the progeny of the original cell which has been transfected.Thus, a “host cell” as used herein generally refers to a cell which hasbeen transfected with an exogenous DNA sequence. Cells from the stablehuman cell line, 293 (readily available through, e.g., the American TypeCulture Collection under Accession Number ATCC CRL1573) can be used. Forexample, the human cell line 293 is a human embryonic kidney cell linethat has been transformed with adenovirus type-5 DNA fragments (Grahamet al. (1977) J. Gen. Virol. 36:59), and expresses the adenoviral E1aand E1b genes (Aiello et al. (1979) Virology 94:460). The 293 cell lineis readily transfected, and provides a convenient platform in which toproduce rAAV virions.

Methods of producing an AAV virion in insect cells are known in the art,and can be used to produce a subject rAAV virion. See, e.g., U.S. PatentPublication No. 2009/0203071; U.S. Pat. No. 7,271,002; and Chen (2008)Mol. Ther. 16:924.

Pharmaceutical Compositions

The present disclosure provides a pharmaceutical composition comprising:a) a subject rAAV virion, as described above; and b) a pharmaceuticallyacceptable carrier, diluent, excipient, or buffer. In some embodiments,the pharmaceutically acceptable carrier, diluent, excipient, or bufferis suitable for use in a human.

Such excipients, carriers, diluents, and buffers include anypharmaceutical agent that can be administered without undue toxicity.Pharmaceutically acceptable excipients include, but are not limited to,liquids such as water, saline, glycerol and ethanol. Pharmaceuticallyacceptable salts can be included therein, for example, mineral acidsalts such as hydrochlorides, hydrobromides, phosphates, sulfates, andthe like; and the salts of organic acids such as acetates, propionates,malonates, benzoates, and the like. Additionally, auxiliary substances,such as wetting or emulsifying agents, pH buffering substances, and thelike, may be present in such vehicles. A wide variety ofpharmaceutically acceptable excipients are known in the art and need notbe discussed in detail herein. Pharmaceutically acceptable excipientshave been amply described in a variety of publications, including, forexample, A. Gennaro (2000) “Remington: The Science and Practice ofPharmacy,” 20th edition, Lippincott, Williams, & Wilkins; PharmaceuticalDosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds.,7^(th) ed., Lippincott, Williams, & Wilkins; and Handbook ofPharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3^(rd) ed.Amer. Pharmaceutical Assoc.

A subject composition can comprise a liquid comprising a subject rAAVvirion in solution, in suspension, or both. As used herein, liquidcompositions include gels. In some cases, the liquid composition isaqueous. In some embodiments, the composition is an in situ gellableaqueous composition, e.g., an in situ gellable aqueous solution. Aqueouscompositions have ophthalmically compatible pH and osmolality.

Methods of Delivering a Gene Product to a Retinal Cell

The present disclosure provides a method of delivering a gene product toa retinal cell (e.g., a Müller cell) in an individual, the methodcomprising administering to the individual a subject rAAV virion asdescribed above. The methods generally involve introducing a subjectrAAV virion into the eye of an individual, where the rAAV virion entersa retinal cell (e.g., a Müller cell) in the eye of the individual, andwhere the gene product encoded by the heterologous polynucleotidepresent in the rAAV virion is produced in the retinal cell. The eye canbe one that has impaired vision and/or that has an ocular disease. Theeye can be one that is at elevated risk of developing impaired visionand/or an ocular disease. Introduction of a subject rAAV virion into theeye of an individual can be carried out by intraocular injection, byintravitreal injection, by intravitreal implant, subretinal injection,suprachoroidal administration, intravenous administration, or by anyother convenient mode or route of administration.

In some cases, a subject rAAV virion, when introduced (e.g., viaintravitreal injection) into an eye of an individual, provides for highlevel production of the heterologous gene product encoded by the rAAV inthe eye. For example, a heterologous polypeptide encoded by the rAAV canbe produced in the eye at a level of from about 1 μg to about 50 μg, orgreater than 50 μg. As another example, a heterologous polypeptideencoded by the rAAV can be produced in the vitreous fluid of the eye ata level of from about 100 pg/mL to about 5000 pg/mL vitreous fluid,e.g., from about 100 pg/mL to about 500 pg/mL, from about 500 pg/mL toabout 1000 pg/mL, from about 1000 pg/mL to about 2000 pg/mL, from about2000 pg/mL to about 3000 pg/mL, from about 3000 pg/mL to about 4000pg/mL, or from about 4000 pg/mL to about 5000 pg/mL. In some cases, apolypeptide encoded by the rAAV can be produced in the vitreous fluid ofthe eye at a level of greater than 5000 pg/mL vitreous fluid.

In some cases, a subject rAAV virion, when introduced (e.g., viaintravitreal injection) into an eye of an individual, provides forproduction of the heterologous gene product encoded by the rAAV in atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 50% at least about 60%, at least about 70%, at least about80%, or more than 80%, of the Müller cells in the eye.

In some embodiments, a subject rAAV virion, when introduced (e.g., viaintravitreal injection) into an eye of an individual, provides forproduction of the heterologous gene product encoded by the rAAV for aperiod of time of from about 2 days to about 6 months, e.g., from about2 days to about 7 days, from about 1 week to about 4 weeks, from about 1month to about 2 months, or from about 2 months to about 6 months. Insome embodiments, a subject rAAV virion, when introduced (e.g., viaintravitreal injection) into an eye of an individual, provides forproduction of the heterologous gene product encoded by the rAAV for aperiod of time of more than 6 months, e.g., from about 6 months to 20years or more, or greater than 1 year, e.g., from about 6 months toabout 1 year, from about 1 year to about 2 years, from about 2 years toabout 5 years, from about 5 years to about 10 years, from about 10 yearsto about 15 years, from about 15 years to about 20 years, or more than20 years.

The gene product can be a polypeptide or a nucleic acid. Nucleic acidgene products include, e.g., an interfering RNA (e.g., an shRNA, ansiRNA, and the like), a ribozyme, an antisense RNA, and an aptamer, asdescribed above.

Where the gene product is an interfering RNA (RNAi), suitable RNAiinclude RNAi that decrease the level of an angiogenic factor in a cell.For example, an RNAi can be an shRNA or siRNA that reduces the level ofVEGF in a cell. RNAi agents that target VEGF include, e.g., an RNAidescribed in U.S. Patent Publication No. 2011/0224282. For example, ansiRNA specific for VEGF-A, VEGFR1, or VEGFR2 would be suitable. Suitablenucleic acid gene products also include a ribozyme specific for VEGF-A,VEGFR1, or VEGFR2; an antisense specific for VEGF-A, VEGFR1, or VEGFR2;siRNA specific for VEGF-A, VEGFR1, or VEGFR2; etc.

Also suitable as a gene product is an miRNA that reduces the level ofVEGF by regulating VEGF gene expression, e.g., throughpost-transcriptional repression or mRNA degradation. Examples ofsuitable miRNA include, e.g., miR-15b, miR-16, miR-20a, and miR-20b.See, e.g., Hua et al. (2006) PLoS ONE 1:e116.

Also suitable is an anti-VEGF aptamer (e.g., EYE001). For anti-VEGFaptamers, see, e.g., Ng et al. (2006) Nature Reviews Drug Discovery5:123; and U.S. Pat. Nos. U.S. Pat. Nos. 6,426,335; 6,168,778;6,147,204; 6,051,698; and 6,011,020. For example, an aptamer directedagainst VEGF₁₆₅, the isoform primarily responsible for pathologicalocular neovascularization and vascular permeability, would be suitable.

Where the gene product is a polypeptide, exemplary polypeptides includeneuroprotective polypeptides and anti-angiogenic polypeptides. Suitablepolypeptides include, but are not limited to, glial derived neurotrophicfactor (GDNF), fibroblast growth factor 2 (FGF-2), nurturin, ciliaryneurotrophic factor (CNTF), nerve growth factor (NGF; e.g., nerve growthfactor-(3), brain derived neurotrophic factor (BDNF), neurotrophin-3(NT-3), neurotrophin-4 (NT-4), neurotrophin-6 (NT-6), epidermal growthfactor (EGF), pigment epithelium derived factor (PEDF), a Wntpolypeptide, and a member of the hedgehog family (sonic hedgehog, indianhedgehog, and desert hedgehog, etc.).

GDNF can be synthesized in cells as a 211-amino acid residueprepropeptide that is processed to yield a dimeric protein composed oftwo 134-amino acid residue subunits. GDNF has been described amply inthe literature; see, e.g., Lin et al. (1993) Science 260:1130; Grimm etal. (1998) Hum. Mol. Genet. 7:1873; Airaksinen and Saarma (2002) NatureReviews 3:383; and Kyuno and Jones (2007) Gene Expr. Patterns 7:313.GDNF amino acid sequences are known; see, e.g., GenBank Accession Nos.NP_000505, NP_001177397; NP_001177398; and NP_954701. Suitable for useherein is a GDNF polypeptide having at least about 85%, at least about90%, at least about 95%, or 100%, amino acid sequence identity to acontiguous stretch of 134 amino acids of the amino acid sequence ofamino acids 78-211 of the sequence set forth in SEQ ID NO:10. Activefragments of GDNF are also suitable for use. In some embodiments, a GDNFpolypeptide has a length of from about 75 amino acids (aa) to about 100aa, from about 100 aa to about 134 aa, from about 134 aa to about 185aa, from about 185 aa to about 202 aa, from about 202 aa to about 211aa, or from about 211 aa to about 228 aa.

PEDF is an approximately 418-amino acid polypeptide that exhibits bothneurotrophic and anti-angiogenic properties. Steele et al. (1993) Proc.Natl. Acad. Sci. USA 90:1526. PEDF amino acid sequences are known; see,e.g., GenBank Accession No. NP_002606; and Steele et al. (1993) supra. Asuitable PEDF polypeptide can have at least about 85%, at least about90%, at least about 95%, or 100%, amino acid sequence identity to acontiguous stretch of from about 25 amino acids (aa) to about 35 aa,from about 35 aa to about 45 aa, from about 45 aa to about 50 aa, fromabout 50 aa to about 100 aa, from about 100 aa to about 200 aa, fromabout 200 aa to about 300 aa, from about 300 aa to about 400 aa, or fromabout 400 aa to 418 aa, of the amino acid sequence set forth in SEQ IDNO:11. In some cases, the PEDF polypeptide is an active fragment. Forexample, a fragment comprising amino acids 24-57 can exhibitanti-angiogenic properties (see, e.g., Amaral and Becerra (2010) Invest.Ophthalmol. Vis. Sci. 51:1318); and a fragment comprising amino acids58-101 can exhibit neurotrophic properties (see, e.g., Filleur et al.(2005) Cancer Res. 65:5144).

Anti-angiogenic polypeptides include, e.g., vascular endothelial growthfactor (VEGF) antagonists. Suitable VEGF antagonists include, but arenot limited to, inhibitors of VEGFR1 tyrosine kinase activity;inhibitors of VEGFR2 tyrosine kinase activity; an antibody to VEGF; anantibody to VEGFR1; an antibody to VEGFR2; a soluble VEGFR; and thelike. See, e.g., Takayama et al. (2000) Cancer Res. 60:2169-2177; Moriet al. (2000) Gene Ther. 7:1027-1033; and Mahasreshti et al. (2001)Clin. Cancer Res. 7:2057-2066; and U.S. Patent Publication No.20030181377. Antibodies specific for VEGF include, e.g., bevacizumab(AVASTIN™) and ranibizumab (also known as rhuFAb V2). Also suitable foruse are anti-angiogenic polypeptides such as endostatin, PEDF, andangiostatin.

Anti-angiogenic polypeptides include, e.g., recombinant polypeptidescomprising VEGF receptors. For example, a suitable anti-angiogenicpolypeptide would be the soluble form of the VEGFR-1, known as sFlt-1(Kendall et al. (1996) Biochem. Biophys. Res. Commun. 226:324). Suitableanti-angiogenic polypeptides include an immunoglobulin-like (Ig) domain2 of a first VEGF receptor (e.g., Flt1), alone or in combination with anIg domain 3 of a second VEGF receptor (e.g., Flk1 or Flt4); theanti-angiogenic polypeptide can also include a stabilization and/or amultimerization component. Such recombinant anti-angiogenic polypeptidesare described in, e.g., U.S. Pat. No. 7,521,049.

Anti-VEGF antibodies that are suitable as heterologous gene productsinclude single chain Fv (scFv) antibodies. See, e.g., U.S. Pat. Nos.7,758,859; and 7,740,844, for anti-VEGF antibodies.

Method of Treating a Retinal Disease

The present disclosure provides a method of treating a retinal disease,the method comprising administering to an individual in need thereof aneffective amount of a subject rAAV virion as described above. A subjectrAAV virion can be administered via intraocular injection, byintravitreal injection, by intravitreal implant, or by any otherconvenient mode or route of administration.

A “therapeutically effective amount” will fall in a relatively broadrange that can be determined through experimentation and/or clinicaltrials. For example, for in vivo injection, e.g., injection directlyinto the eye, a therapeutically effective dose will be on the order offrom about 10⁶ to about 10¹⁵ of the rAAV virions, e.g., from about 10⁸to 10¹² rAAV virions. For example, for in vivo injection, e.g.,injection directly into the eye, a therapeutically effective dose willbe on the order of from about 10⁶ to about 10¹⁵ infectious units, e.g.,from about 10⁸ to about 10¹² infectious units. Other effective dosagescan be readily established by one of ordinary skill in the art throughroutine trials establishing dose response curves.

In some cases, a therapeutically effective amount of a subject rAAVvirion is an amount that, when administered to an individual (e.g.,administered via intravitreal injection into an eye (e.g., a visuallyimpaired eye; an eye having an ocular disease; an eye that is at risk ofdeveloping an ocular disease) of the individual) in one or more doses,is effective to slow the progression of retinal degeneration in theindividual. For example, a therapeutically effective amount of a subjectrAAV virion can be an amount that, when administered to an individual(e.g., administered via intravitreal injection to an individual) in oneor more doses, is effective to slow the progression of retinaldegeneration by at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, or more than80%, compared to the progression of retinal degeneration in the absenceof treatment with the rAAV virion.

In some cases, a therapeutically effective amount of a subject rAAVvirion is an amount that, when administered to an individual (e.g.,administered via intravitreal injection into an eye of the individual)in one or more doses, is effective to improve vision in the individual.For example, a therapeutically effective amount of a subject rAAV virioncan be an amount that, when administered to an individual (e.g.,administered via intravitreal injection into an eye of the individual)in one or more doses, is effective to improve vision by at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, or more than 80%, compared to the individual'svision in the absence of treatment with the rAAV virion.

In some cases, a therapeutically effective amount of a subject rAAVvirion is an amount that, when administered to an individual (e.g.,administered via intravitreal injection into an eye of the individual)in one or more doses, is effective to decrease the rate of vision lossin an eye with impaired vision.

Improvement of clinical symptoms are monitored by one or more methodsknown to the art, for example, tests of functional vision, such asvisual acuity, visual field, contrast sensitivity, color vision,mobility, and light sensitivity. Clinical symptoms may also be monitoredby anatomical or physiological means, such as indirect ophthalmoscopy,fundus photography, fluorescein angiopathy, optical coherencetomography, electroretinography (full-field, multifocal, or other),external eye examination, slit lamp biomicroscopy, applanationtonometry, pachymetry, autorefaction, or other measures of functionalvision.

Multiple doses of a subject rAAV virion can be administered to anindividual in need thereof. Where multiple doses are administered over aperiod of time, an active agent is administered once a month to aboutonce a year, from about once a year to once every 2 years, from aboutonce every 2 years to once every 5 years, or from about once every 5years to about once every 10 years, over a period of time. For example,a subject rAAV virion is administered over a period of from about 3months to about 2 years, from about 2 years to about 5 years, from about5 years to about 10 years, from about 10 years to about 20 years, ormore than 20 years. The actual frequency of administration, and theactual duration of treatment, depends on various factors.

As an example, a subject method of treating an ocular disorder caninclude administering an initial dose of a subject rAAV virion; andadministering at least a second dose (a subsequent dose) of the rAAVvirion. Where two or more subsequent doses are administered, thesubsequent dose(s) can be separated in time from each other by at leastone month, at least 3 to 6 months, at least 6 months to 1 year, at least1 year to 5 years, at least 5 years to 10 years, at least 10 years to 20years, or more than 20 years.

Ocular diseases that can be treated or prevented using a subject methodinclude, but are not limited to, selected from acute macularneuroretinopathy; macular telangiectasia; Behcet's disease; choroidalneovascularization; diabetic uveitis; histoplasmosis; maculardegeneration, such as acute macular degeneration, Scorsby's maculardystrophy, early or intermediate (dry) macular degeneration, or a formof advanced macular degeneration, such as exudative macular degenerationor geographic atrophy; edema, such as macular edema, cystoid macularedema and diabetic macular edema; multifocal choroiditis; ocular traumawhich affects a posterior ocular site or location; ocular tumors;retinal disorders, such as central retinal vein occlusion, diabeticretinopathy (including proliferative and non-proliferative diabeticretinopathy), proliferative vitreoretinopathy (PVR), retinal arterialocclusive disease, retinal detachment, uveitic retinal disease;sympathetic opthalmia; Vogt Koyanagi-Harada (VKH) syndrome; uvealdiffusion; a posterior ocular condition caused by or influenced by anocular laser treatment; posterior ocular conditions caused by orinfluenced by a photodynamic therapy, photocoagulation, radiationretinopathy; epiretinal membrane disorders; central or branch retinalvein occlusion; anterior ischemic optic neuropathy, non-retinopathydiabetic retinal dysfunction; retinitis pigmentosa; retinoschisis; andglaucoma.

Subjects Suitable for Treatment

Subjects suitable for treatment according to a method of the presentdisclosure include individuals having an ocular disease, as describedabove. Subjects suitable for treatment also include individuals atincreased risk (e.g., at increased risk relative to the generalpopulation) of developing an ocular disease. Ocular diseases includethose listed above.

Nucleic Acids and Host Cells

The present disclosure provides an isolated nucleic acid comprising anucleotide sequence that encodes a variant adeno-associated virus (AAV)capsid protein, where the variant AAV capsid protein comprises an aminoacid sequence having at least about 85% at least about 90%, at leastabout 95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the ShH10 amino acid sequence depicted in FIGS.8A-C, where the AAV capsid protein does not comprise an amino acidsequence present in a naturally occurring AAV capsid protein, and wherethe variant capsid protein, when present in an AAV virion, provides forincreased infectivity of the AAV virion for a retinal cell.

The present invention further provides host cells, e.g., isolated(genetically modified) host cells, comprising a subject nucleic acid. Asubject host cell can be an isolated cell, e.g., a cell in in vitroculture. A subject host cell is useful for producing a subject rAAVvirion, as described below. Where a subject host cell is used to producea subject rAAV virion, it is referred to as a “packaging cell.” In someembodiments, a subject host cell is stably genetically modified with asubject nucleic acid. In other embodiments, a subject host cell istransiently genetically modified with a subject nucleic acid.

A subject nucleic acid is introduced stably or transiently into a hostcell, using established techniques, including, but not limited to,electroporation, calcium phosphate precipitation, liposome-mediatedtransfection, baculovirus infection, and the like. For stabletransformation, a subject nucleic acid will generally further include aselectable marker, e.g., any of several well-known selectable markerssuch as neomycin resistance, and the like.

A subject host cell is generated by introducing a subject nucleic acidinto any of a variety of cells, e.g., mammalian cells, including, e.g.,murine cells, and primate cells (e.g., human cells). Suitable mammaliancells include, but are not limited to, primary cells and cell lines,where suitable cell lines include, but are not limited to, HeLa cells(e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells(e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No.CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721),COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells(ATCC No. CCLI.3), Sf9 cells, human embryonic kidney (HEK) cells (ATCCNo. CRL1573), HLHepG2 cells, and the like.

In some embodiments, a subject genetically modified host cell includes,in addition to a nucleic acid comprising a nucleotide sequence encodinga variant AAV capsid protein, as described above, a nucleic acid thatcomprises a nucleotide sequence encoding one or more AAV rep proteins.In other embodiments, a subject host cell further comprises an rAAVvector. An rAAV virion can be generated using a subject host cell.Methods of generating an rAAV virion are described in, e.g., U.S. PatentPublication No. 2005/0053922 and U.S. Patent Publication No.2009/0202490.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1: Generation of rAAV Virions with Variant Capsids andExhibiting Increased Infectivity of Müller Glial Cells Materials andMethods

Generation of rAAV Vectors

Vectors were produced by the plasmid co-transfection method (Koerber etal. (2009) Mol. Ther. 17:2088), and the resulting lysates were purifiedvia iodixanol gradient ultracentrifugation as previously described.Koerber et al. Mol Ther. 2008:16:1703-1709. This fraction was thenpassed through a heparin column, which was washed with 5 mLphosphate-buffered saline (PBS) and eluted with 5 mL of a 1 M NaClsolution. The resulting viral fractions were desalted and concentratedwith Amicon Ultra-15 Centrifugal Filter Units to a final volume of 200Vector was then titered for DNase-resistant vector genomes by real timepolymerase chain reaction (PCR) relative to a standard.

Intraocular Administration Routes

Adult wild type Sprague Dawley rats were used for the studies discussedin this Example. All animal procedures were conducted according to theARVO Statement for the Use of Animals and the guidelines of the Officeof Laboratory Animal Care at the University of California, Berkeley.Before vector administration, rats were anesthetized with ketamine (72mg/kg) and xylazine (64 mg/kg) by intraperitoneal injection. Anultrafine 30½-gauge disposable needle was passed through the sclera, atthe equator and next to the limbus, into the vitreous cavity. Injectionof 5 μl, containing 1-5×10¹² vg/ml of AAV dsCAG-green fluorescentprotein (GFP), was made with direct observation of the needle in thecenter of the vitreous cavity.

Fundus Photography

Fundus imaging was performed one to eight weeks after injection with afundus camera (Retcam II; Clarity Medical Systems Inc., Pleasanton,Calif.) equipped with a wide angle 130° retinopathy of prematurity (ROP)lens to monitor eGFP expression in live, anesthetized rats. Pupils weredilated for fundus imaging with phenylephrine (2.5%) and atropinesulfate (1%).

Cryosections

One to eight weeks after vector injection, rats were humanelyeuthanized, the eyes were enucleated, a hole was introduced in thecornea, and tissue was fixed with 10% neutral buffered formalin for 2-3hours. The cornea and lens were removed. The eyecups were washed in PBSfollowed by 30% sucrose in the same buffer overnight. Eyes were thenembedded in optimal cutting temperature embedding compound (OCT; MilesDiagnostics, Elkhart, Ind.) and oriented for 10 μm thick transverseretinal sections.

Immunolabeling and Histological Analysis

Tissue sections were rehydrated in PBS for 5 min, followed by incubationin a blocking solution of 1% bovine serum albumin (BSA), 0.5% TritonX-100, and 2% normal donkey serum in PBS for 2-3 hours. Slides were thenincubated with commercial monoclonal antibodies raised against glutaminesynthetase in rabbit (Sigma G2781) at a 1:3000 dilution, calbindin(Abcam ab11426-50) in rabbit at a 1:1000 dilution, vimentin in mouse(Dako M0725) at a 1:1000 dilution, or laminin in rabbit (Sigma, L9393)at 1:100 in blocking solution, overnight at 4° C.

The sections were then incubated with Cy3-conjugated secondaryanti-rabbit or anti-mouse antibody (Molecular Probes) at a 1:1000dilution in blocking solution for 2 hours at room temperature. Theresults were examined by fluorescence microscopy using an Axiophotmicroscope (Zeiss, Thornwood, N.Y.) equipped with a Xcite PC200 lightsource and QCapturePro camera or a confocal microscope (LSMS; Carl ZeissMicroimaging). Transduction profiles were analyzed by countingindividual cells from whole retinas in 15 μm cryosections (n=6) usingfluorescence microscopy. Efficiencies were calculated by dividing thetotal number of these transduced Müller cells by the total number ofMüller cells in the retinal slice (mm) in which these cells were present(n=6).

In Vitro Transduction Analysis

Transduction studies using rAAV CMV-GFP (GFP operably linked to acytomegalovirus promoter) were performed with 5×10⁴ cells (CHO, pgsA,Pro5, and Lec1) in 12-well plates. Cells were transduced with rAAV GFPvectors at a genomic multiplicity of infection (gMOI) of 10³-10⁵ (n=3),and the percentage of GFP-expressing cells was determined by flowcytometry 48 hours post-infection.

Results In Vivo Characterization of Müller Cell Permissive Variants

Several novel AAV capsids were recently evolved, that efficientlytransduced both primary human astrocytes in vitro and rat astrocytes invivo using highly diverse AAV libraries (>10⁷). Koerber et al. (2009)supra. These variants were generated via multiple evolutionary rounds(i.e. diversification followed by positive selection for enhancedastrocyte transduction in vitro) with several distinct libraries: (1) anAAV2 random mutagenesis library generated via error prone PCR (Maheshriet al. Nat Biotechnol. 2006; 24:198-2.04), (2) a random chimera AAVlibrary generated by shuffling the cap genes of 7 natural human andnon-human AAV serotypes (Koerber et al. Mol Ther. 2008; 16:1703-1709),and (3) a novel AAV2 library with surface-exposed loops of the capsidlibrary diversified based on a bioinformatics approach. Koerber et al.(2009) supra.

The utility of these variants was explored for intravitreal transductionof Müller cells. Here, the eight isolated mutants that demonstrated thegreatest in vitro astrocyte infectivity (FIG. 8) were individuallyanalyzed for the ability to transduce the retina from the vitreous usingdouble-stranded (ds) AAV CAG-GFP vectors purified via iodixanol gradientultracentrifugation and heparin affinity chromatography. Intravitrealinjections of 2.5×10¹⁰ genomic particles revealed one previouslyunreported variant named ShH10, derived from an AAV6 parent serotypefrom the shuffled (ShH) library, that showed a dramatic increase inspecificity and efficiency for Müller cells relative to controls atthree weeks post-injection (FIGS. 2 and 3). Interestingly, no othermutants demonstrated visible expression as determined by GFP fundusimaging and immunohistochemistry. Recombinant ShH10 (rShH10) led todiffuse expression throughout the retina with a highly specifictransduction profile of approximately 94% Müller cells, 2% interneurons,and 4% retinal ganglion cells (FIGS. 2, 3, 4). In comparison, the parentvector, AAV6, showed very low transduction of the retina, and therelated AAV2 vector showed a less specific retinal tropism with atransduction profile of approximately 76% Müller cells, 3% interneurons,and 21% retinal ganglion cells (FIGS. 2, 3, 4). Quantification oftransduction efficiencies revealed that ShH10 was approximately 62% moreefficient at infecting Müller cells relative to AAV2, infecting 22% vs.14% of total Müller cells respectively in transverse retinal slices(FIG. 3B).

Temporal observation of ShH10 expression using fundus imaging, coupledwith anti-laminin immunostaining of retinal flatmounts to visualizevasculature, also revealed a unique tropism for retinal astrocytes atearlier time points following injection (FIG. 5). Unlike Müller cells,retinal astrocytes are not derived from the retinal neuroepithelium, butserve some analogous roles in the retina including providing nutritionalsupport to neurons, neurotransmitter metabolism, and ionic homeostasis.Trivino et al. Vision Res. 1996; 36:2015-2028. They also serve as axonalglial sheaths for ganglion cells bodies and envelop the retinalvasculature, forming part of the blood-brain barrier. One weekpost-injection, fundus imaging revealed localized expression near thoseareas dense in retinal astrocytes, e.g. the optic nerve and alongretinal vasculature (FIG. 5a )). Additionally, transverse retinalsections showed that areas underlying major vasculature bore strongMüller expression (FIG. 5d ). At later time points (2-3 weeks),expression became more evenly spread, but interestingly, those regionsin proximity to vasculature ultimately maintained the strongest Müllercell expression (FIG. 4)).

FIG. 1 depicts Müller glia in the retina. Illustration of Müller gliaspanning the entire retina, where they ensheath all neuronal types fromthe RGC (bottom) to the photoreceptors. Modified from Histology of theHuman Eye, an Atlas and Textbook. Hogan, Michael J., Jorge A. Alvarado,Joan Esperson Weddell. Philadelphia: W. B. Saunders, 1971.

FIGS. 2A-I depict rShH10 expression following intravitreal injection inthe adult rat retina. Confocal imaging of immunostained transverseretinal sections 3 weeks post-injection of 2.5×10¹⁰ viral particles(vector genomes) of dsCAG-GFP vectors with capsids from AAV2 (A-C),ShH10 (D-F), and AAV6 (G-I) (n=6). Glutamine synthetase (GS) staining(red) (B,E,H) and visualization of colocalization (C,F,I) reveals morerobust Müller cell expression by ShH10 (E, F) relative to AAV2 (B,C),whereas AAV6 shows no visible expression (G-I). Additionally, GFPexpression shows specific transduction of Müller cells by ShH10 (D)compared to AAV2 (A), which exhibits considerably more transduction ofretinal ganglion cells and interneurons.

FIGS. 3A and 3B depict transduction specificity and efficiency of ShH10.Representative retinal slices from injected eyes were quantified for thenumber of each cell type that was infected, as determined via GFPexpression, to generate histograms comparing tropism profiles (A) andMüller transduction efficiencies (B) of rAAV2, rShH10, and rAAV6dsCAG-GFP. Transduction efficiencies were calculated based on the ratioof Müller cells infected relative to the total number of Müller cells ina 10 μm transverse retinal slice (n=6). Error bars represent standarddeviation among sample population.

FIGS. 4A and 4B depict rShH10 expression in the whole retina followingintravitreal injection. Fluorescence microscopy of transverse retinalsections from rShH10 CAG-GFP and rAAV6 CAG-GFP injected animals 3 weekspost-injection reveals broadly spread expression by ShH10 (B), with themost prominent expression localized at the injection site. AAV6 (A)shows no visible expression.

FIGS. 5A-D depict retinal astrocyte infectivity of ShH10. Fundus imagingof rShH10 CAG-GFP injected animals at one week (A) reveals acharacteristic expression pattern localized near major vasculature andthe optic nerve, which subsequently shows spreading after three weeks(B). Closer examination by flatmount (C) through laminin (red) and DAPI(blue) staining reveals a strong localization of GFP expression alongthe edges of retinal blood vessels, areas dense in retinal astrocytes.Transverse sections (D) stained for calbindin (red), a marker of RGCsand interneurons in the retina, illustrate a local region of expressionwithin retinal astrocytes and Müller cells ensheathing a blood vessel.

Mutational Analysis of ShH10

ShH10 is highly Müller cell selective, while AAV6 yields no detectableretinal expression upon intravitreal administration, yet ShH10 differsfrom AAV6 at only four residues: I319V, N451D, D532N, and H642N (FIG.8)). To analyze the contributions of each of these mutations to ShH10'snovel phenotype, single point mutants and all potential double mutantswere generated from the AAV6 cap gene via site-directed mutagenesis.Each resulting variant was used to package rAAV-CMV-GFP and was purifiedvia iodixanol gradient ultracentrifugation. To characterize the in vitroinfectivity of these mutants, and in particular their glycan dependencein light of the substantial role proteoglycans and glycoproteins play inAAV transduction (Goncalves Wad J. 2005; 2:43; Xie et al. Proc Natl AcadSci USA. 2002; 99:10405-10410; Wu et al. J Virol. 2006; 80:9093-9103),their relative transduction efficiencies were analyzed on a panel ofcell types: Pro5, a Pro5 mutant (Lec1) deficient in N-linked sialicacid, CHO, and a CHO derivative (pgsA) deficient in allglycosaminoglycans. Bane et al. J Biol Chem. 1991; 266; 10287-10293.AAV6 exhibited a dependence on N-linked sialic acids for efficienttransduction, as previous studies have indicated (FIG. 6c ). Wu et al.(2006) supra. However, the N451D mutation decreased the viral dependenceon N-linked sialic acids, and the D532N mutation increased the viraltransduction in the absence of N-linked sialic acids (FIG. 6c )). ThisD532N mutation, located near the HSPG binding domain of the AAV6 capsid(Wu et al. J Virol. 2006; 80:11393-11397), may enable the virus toutilize a transduction pathway distinct from AAV6 (FIG. 7).

Whereas AAV6 does not utilize HSPG for transduction (FIG. 6b ) (Wu etal. J Virol. 2006; 80:9093-9103), several of the SHIM mutations confer anew dependence on HSPG. Intriguingly, AAV6 N451D exhibited lowertransduction levels relative to AAV6 in CHO cells, but when coupled withthe D532N mutation was more infective than either AAV6 or AAV6 D532N(FIG. 6a ). Comparing infection efficiencies among the single pointmutants between CHO and pgsA cells, cell lines containing and lackingHSPG respectively, AAV6 D532N was the only mutant to exhibit asubstantial HSPG dependence, which became more pronounced when coupledwith mutations I319V and N451D (FIG. 6b ). The enhanced infectivity ofSHIM is thus likely due to a synergy between mutations that in partaugments HSPG affinity as suggested by the heparin affinity chromatogram(FIG. 9). To determine whether AAV6 mutations that enhance infectivityalso function in vivo, equal titer intravitreal injections of 5×10°genomic particles of recombinant vector mutants carrying dsCAG-GFPrevealed that that only AAV6 N451D was sufficient to confer theintravitreal Müller tropism. This mutant was considerably more efficientthan AAV6 on Müller cells, though only half as efficient as ShH10 (FIGS.3 and 7 b).

FIGS. 6A-C depict in vitro characterization of ShH10. (A) CHO celltransduction by rAAV6, rAAV6 N451D, rAAV6 D532N, and rAAV6 N451D+D532Ncarrying CMV-GFP. (B) CHO/PgsA transduction demonstrating the HSPGdependence of various permutations of the mutations that comprise ShH10.(C) Pro5/Lec1 transduction examining sialic acid dependence of variouspermutations of the mutations that compose ShH10.

FIGS. 7A-C depict rAAV6 N451D expression following intravitrealinjection. Confocal imaging of immunostained transverse retinal sections3 weeks post-injection of rAAV6 N451D CAG-GFP (B, C). GFP expressionanalysis (B) and overlay with GS (C) reveal this mutant to be sufficientfor an intravitreal Müller infection, though at a reduced efficiencyrelative to ShH10 (FIG. 1 D, F). Mapping of this mutation onto the AAV6capsid subunit VP3 (A) (blue) shows its location near the three-foldaxis of symmetry of the assembled capsid. Three-dimensional models ofthe AAV6 VP3 subunit were generated using Swiss Model with thecoordinates of AAV2 (Protein Databank accession no. 1LP3) supplied as atemplate and images were rendered in Pymol and Rasmol. Additionally, theD532N mutation (green) maps near the HSPG-binding domain (purple).

FIGS. 8A-C depict amino acid sequences of wild-type and variant AAVcapsids.

FIG. 9 depicts the elution profile from a heparin column for ShH10,AAV2, and AAV6. The Y-axis values represent the fraction of viruseluted, and the X-axis represents the concentration of NaCl in theeluant (mM).

Example 2: AAV-Mediated GDNF Secretion from Retinal Glia Slows RetinalDegeneration Materials and Methods:

Generation of rAAV Vectors:

AAV vectors were produced by the plasmid co transfection method. Griegeret al. (2006) Nat Protoc 1: 1412-1428, rAAV was purified via iodixanolgradient ultracentrifugation (Dalkara, D, et al. (2009) Mol Ther 17:2096-2102) and heparin column chromatography (GE Healthcare, ChalfontSt. Giles, UK). The viral eluent was desalted and concentrated withAmicon Ultra-15 Centrifugal Filter Units to a final volume of 200 μl andtitered by quantitative polymerase chain reaction (qPCR) relative tostandards.

Intraocular Injections:

TgS334-4ter rats were used for all studies, and all animal procedureswere conducted according to the ARVO Statement for the Use of Animalsand the guidelines of the Office of Laboratory Animal Care at theUniversity of California, Berkeley. Rats were first anesthetized withketamine (72 mg/kg) and xylazine (64 mg/kg) by intraperitonealinjection. An ultrafine 30½-gauge disposable needle was then passedthrough the sclera, at the equator and next to the limbus, into thevitreous cavity. Five μl, containing 1-5×10¹² vg/ml of AAV were injectedwith direct observation of the needle in the center of the vitreouscavity.

Cryosections:

Animals were humanely euthanized by CO₂ overdose and cervicaldislocation. Eyes were enucleated and immersion fixed in 10% formalin.The cornea and lens were removed and the resulting eye-cups werecryoprotected in 30% sucrose before embedding in OCT compound (MilesDiagnostics, Elkhart, Ind.). 5-10 μm thick transverse retinal sectionswere cut.

Immunolabeling:

Tissue sections re blocked in 1% BSA, 0.5% Triton X-100, and 2% normaldonkey serum for 2-3 hours and treated with a rabbit anti-glutaminesynthetase monoclonal antibody (Sigma G2781) at a 1:3000 dilution inblocking solution overnight at 4° C. After 3 PBS washes, Cy3-conjugatedanti-rabbit secondary (GE Healthcare) was applied at a 1:1000 dilutionin blocking solution for 2 hours at room temperature. The results wereexamined by confocal microscopy (LSMS; Carl Zeiss Microimaging).

Electroretinography:

Rats were dark-adapted for minimum of 2 hours and then anesthetized,followed by pupil dilation. Contact lenses were positioned on the corneaof both eyes. Reference electrodes were inserted subcutaneously in thecheeks and a ground electrode was inserted in the tail.Electroretinograms were recorded (Espion ERG system; Diagnosys LLC,Littleton, Mass.) in response to seven light flash intensities rangingfrom −4 to 1 log cd*s/m². Each stimulus was presented in series ofthree. Light flash intensity and timing were computer controlled. Datawere analyzed with MatLab (v7.7; Mathworks, Natick, Mass.). ERG a and bwaves from control and treated eyes were compared using Mann-Whitneypaired t test.

Histology:

Rats were euthanized by CO₂ overdose. The superior cornea was marked,and enucleated eyes were immersion fixed in formalin followed by removalof cornea and lens. Eye cups were then fixed in 1% osmium tetroxide,dehydrated by incubation in increasing ethanol concentrations and afinal incubation in 100% propylene oxide. The samples were then embeddedin an epon-araldite resin and hardened overnight at 65° C. One μm thinplastic sections were cut along the vertical meridian, through the opticnerve with a sapphire blade. Measurements of ONL, IPL and OS thicknessfrom the optic nerve head (ONH) to ora serrata in 3 rats 3 months posttreatment were made on high resolution montages of the retinas imaged at40× using ImageJ software. Fifty four measurements of the ONL, IPL andOS were made at 18 contiguous fields around the entire retinal section(3 measurements per field). These measurements were plotted as adistribution of thickness across the central retina.

ELISA:

Brief sonication was used to homogenize treated and control retinas.ELISA was performed using the DuoSet Kit for human GDNF (R&D systems)according to the manufacturer's instructions.

Results

ShH10 leads to selective and efficient targeting of Müller glia in a ratmodel of RP.

As described in Example 1, the engineered AAV variant ShH10 had revealedefficient and specific transduction of rat Müller glia in wild-typeanimals. Klimczak, et al. (2009) PLoS One 4: e7467. Since high-levelGDNF expression is of interest, the infectivity of ShH10 was furtherenhanced by mutating a surface exposed tyrosine residue to phenylalaninefor potentially more efficient trafficking to the nucleus. Petrs-Silva,H, et al. (2009) Mol Ther 17: 463-471; and Zhong, L, et al. (2008) ProcNatl Acad Sci USA 105: 7827-7832. Intravitreal injection of thisrecombinant ShH10.Y445F variant with a scCAG.GFP transgene in 5334-4terrats revealed strong, selective expression in Müller cells throughoutthe retina, peaking 3 weeks post injection (FIG. 10a-e ). Furthermore,53% of all Müllner cells showed GFP expression in TgS334-ter retinasinfected with ShH10.Y445F. This indicates a significant increasecompared to the number of Müller cells infected in wild type retinasafter ShH10 vector introduction. Klimczak, et al. (2009) PLoS One 4:e7467. This is likely due to an increased transduction efficiencyafforded by the additional tyrosine mutation, alongside the morepermissive nature of degenerating retinas to AAV mediated transduction.Kolstad, et al. (2010) Hum Gene Ther 21: 571-578.

FIGS. 10A-E. ShH10.Y445F.scCAG-GFP drives strong pan-retinal expressionin Müller cells when intravitreally injected into S334-4ter rat eyes.(a) Representative fundus images at 1 to 4 weeks post injection into p15S334-ter rat eyes show rapid onset of expression at 1 week postinjection before peaking and stabilizing at 3-4 weeks. (b) Highresolution montage of a retinal cryosection through the ONH showing theextent of GFP expression in Müller cells throughout the retina. (c) Lowmagnification (10×) images at 4 representative regions of the retinalcryo section. Left hand panels show GFP expression in Müller cells whileright hand panels also show glutamine synthase staining (in red) andnuclei stained with DAPI in blue. (d) High magnification image (40×)showing selective GFP expression in Müller cells in green alongside anoverlay of GFP with glutamine synthase-staining (red) and DAPI-staining(blue).

Given the high specificity of the vector, a self-complementary hGDNFtransgene driven from the same promoter, was inserted. Yokoi, K, et al.(2007) Invest Ophthalmol Vis Sci 48: 3324-3328; and McCarty, D M (2008)Mol Ther 16: 1648-1656. Following intravitreal delivery of ShH10.Y444FscCAG.hGDNF, enzyme-linked immunosorbent assay (ELISA) measurementsrevealed robust secretion of hGDNF from Müller cells both two and threemonths post injection (FIG. 11). At more than 2.5 ng/mL, these hGDNFlevels are nearly 10-fold higher than those produced in previous studiesthat have achieved photoreceptor degeneration rescue through GDNFoverexpression from retinal neurons after subretinal injection (Frasson,M, et al. (1999) Invest Ophthalmol Vis Sci 40: 2724-2734; McGee et al.(2001) Mol Ther 4: 622-629) or from intraocularly-placed mouse embryonicstem cells (Gregory-Evans, et al. (2009) Mol Vis 15: 962-973).Importantly, the expression is sustained, as ELISA measurements of GDNFin the vitreous of rats 5 months post injection show elevated levels ofthe therapeutic protein, which are both safe (Wu, et al. (2005) Curr EyeRes 30: 715-722) and necessary for sustained rescue.

FIG. 11. ELISA measurements of hGDNF protein in retinal homogenates two(n=7) and three months (n=6) following intravitreal delivery ofShH10.Y444F.scCAG.hGDNF. All animals received hGDNF vector treatment inthe right eye and no injection in the left eye.

Müller Cell Secretion of GDNF Slows Down Retinal Degeneration inS334-4Ter Rats

Electroretinography (ERG) was used to assess the visual function ofS334-4ter animals injected intravitreally at P15 withShH10.Y445F.scCAG-GDNF. At one month post-injection, small increaseswere seen in both a and b-wave amplitudes of the treated eyes relativeto untreated eyes (either uninjected or injected withShH10.Y444F.scCAG.GFP) (FIG. 12a -b, e). Although the ERG amplitudeswere heterogeneous among animals, rescue was consistent between thecontrol and contralateral GDNF-injected eyes, with an average b-wavevalue of 644 μV (+/−142 μV) in treated eyes versus 481 μV (+/−82 μV) forthe control eyes (Figure S3a). Wild-type animals demonstrated a-waveamplitudes of approximately 455 μV (FIG. 12a ) and b-wave amplitudes of1370 μV at the same age (FIG. 12b ). Remarkably, from 3 to 5 monthsafter the injection, the physiological rescue became more pronounced,with an average amelioration of 50% in b-wave amplitude amongst allanimals at 5 months and a nearly two-fold increase observed in oneanimal (FIG. 12d ), with similar improvements in a-wave amplitudes (FIG.12c ). Representative ERG traces corresponding to the average values atone (FIG. 12e ) and five (FIG. 12f ) months post injection are shownbelow.

FIGS. 12A-F. Scatter plots of ERG a (a) and b-wave (b) amplitudes inresponse to 1 log cd*s/m2 in GDNF-treated and contralateral control eyes1 month post-injection and at 5 months (c, d). All animals (n=6) wereinjected at p15. Representative ERG traces at 1 log cd*s/m2 from a wildtype animals eyes (gray solid and dotted traces) and an animal withGDNF-treated (solid black line) versus control eye (dotted black line)at 1 month post-injection (e). Representative ERG trace at 1 log cd*s/m2from a GDNF-treated animal (solid black line) versus contralateralcontrol eye (dotted black line) at 5 months (f).

Histological Rescue of Photoreceptors

Histological rescue was determined by measuring the thickness of theouter nuclear layer (ONL), a well-established indicator of photoreceptorsurvival. Histological examination of GDNF-injected and control retinascorroborate the preservation of function observed in theelectroretinograms. The ONLs of superior and inferior GDNF-treatedretinas were thicker up to 4 mm from the optic nerve head at theinferior retina and up to 2 mm from the optic nerve at the superiorretinas at 3 months post-injection (FIG. 13a ). Additionally, the innerplexiform layer and the photoreceptor outer segments were shorter inmost of the inferior and a fraction of the superior control retinas(FIG. 13a-c ).

FIGS. 13A-G. Measurements of outer nuclear layer thickness (a) innerplexiform layer thickness (b) and photoreceptor outer segment length (c)along the vertical meridian of the eye from the optic nerve head (ONH)to the ora serrata in rats at 3 months post injection (p105). Rats wereeither uninjected (full circles) or injected with ShH10.Y445F.scCAG.GDNF(squares) at p15. Light micrographs from inferior retinas ofShH10.Y445F.scCAG.GDNF injected (d) of uninjected (e) rats at 20×magnification, scale bars are 25 μm. High magnification micrographs frominferior retinas of ShH10.Y445F.scCAG.GDNF injected (e) or uninjected(f) rats at 40× magnification, showing differences in outer segmentslength in the central part of inferior retinas.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1.-30. (canceled)
 31. A recombinant adeno-associated virus (rAAV) virioncomprising: a) a variant AAV capsid protein, wherein the variant AAVcapsid protein comprises an amino acid substitution at amino acid 451 ofthe AAV6 capsid sequence as set forth in SEQ ID NO:1, or thecorresponding position in another AAV parental serotype, wherein the AAVcapsid protein does not comprise an amino acid sequence present in anaturally occurring AAV capsid protein; and b) a heterologous nucleicacid comprising a nucleotide sequence encoding a heterologous geneproduct.
 32. The rAAV virion of claim 31, wherein heterologous geneproduct is a nucleic acid gene product.
 33. The rAAV virion of claim 32,wherein the nucleic acid gene product is an interfering RNA, a ribozyme,an antisense nucleic acid, or an aptamer.
 34. The rAAV virion of claim33, wherein the interfering RNA or the aptamer reduces the level of anangiogenic factor in the retinal cell.
 35. The rAAV virion of claim 31,wherein the heterologous gene product is a polypeptide.
 36. The rAAVvirion of claim 35, wherein the polypeptide is a neuroprotectivepolypeptide.
 37. The rAAV virion of claim 35, wherein the polypeptide isglial derived neurotrophic factor, fibroblast growth factor 2, nurturin,ciliary neurotrophic factor, nerve growth factor, brain derivedneurotrophic factor, epidermal growth factor, a soluble vascularendothelial growth factor (VEGF) receptor, an anti-VEGF antibody, orSonic hedgehog.
 38. The rAAV virion of claim 35, wherein the polypeptideis an anti-angiogenic polypeptide.
 39. The rAAV virion of claim 31,wherein the parental AAV capsid protein is wild-type AAV6 capsidprotein.
 40. The rAAV virion of claim 31, wherein the variant AAV capsidprotein comprises an amino acid change at AAV6 position 532 or thecorresponding position in another AAV parental serotype.
 41. The rAAVvirion of claim 31, wherein the amino acid substitution at amino acid451 of AAV6, or the corresponding position in another AAV parentalserotype, is an aspartic acid.
 42. The rAAV virion of claim 31, whereinthe variant capsid protein comprises from 1 to 10 amino acid differencescompared to a wild-type AAV capsid protein.
 43. A pharmaceuticalcomposition comprising: a) a recombinant adeno-associated virus (rAAV)virion according to claim 31; and b) a pharmaceutically acceptablecarrier, diluent, excipient, or buffer.
 44. A method of delivering agene product to a retinal cell in an individual, the method comprisingadministering to the individual a recombinant adeno-associated virus(rAAV) virion according to claim
 31. 45. The method of claim 44, whereinthe gene product is a polypeptide.
 46. The method of claim 45, whereinthe polypeptide is a neuroprotective factor or an anti-angiogenicfactor.
 47. The method of claim 45, wherein the polypeptide is glialderived neurotrophic factor, fibroblast growth factor 2, nurturin,ciliary neurotrophic factor, nerve growth factor, brain derivedneurotrophic factor, epidermal growth factor, a soluble vascularendothelial growth factor (VEGF) receptor, an anti-VEGF antibody, orSonic hedgehog.
 48. The method of claim 44, wherein the gene product isa nucleic acid.
 49. A method of treating a retinal disease, the methodcomprising administering to an individual in need thereof an effectiveamount of a recombinant adeno-associated virus (rAAV) virion accordingto claim
 31. 50. The method of claim 49, wherein said administering isby intraocular injection.
 51. The method of claim 49, wherein saidadministering is by intravitreal injection.